An Alternative Strategy To Determine the Mitochondrial Proteome Using Sucrose Gradient Fractionation and 1D PAGE on Highly Purified Human Heart Mitochondria Steven W. Taylor,*,† Dale E. Warnock,† Gary M. Glenn,† Bing Zhang,† Eoin Fahy,† Sara P. Gaucher,‡ Roderick A. Capaldi,§ Bradford W. Gibson,‡ and Soumitra S. Ghosh† MitoKor, 11494 Sorrento Valley Road, San Diego California 92121, Institute for Molecular Biology, University of Oregon, Eugene, Oregon 97403, and Buck Institute for Age Research, Novato, California 94945 Received May 22, 2002
An alternative strategy for mitochondrial proteomics is described that is complementary to previous investigations using 2D PAGE techniques. The strategy involves (a) obtaining highly purified preparations of human heart mitochondria using metrizamide gradients to remove cytosolic and other subcellular contaminant proteins; (b) separation of mitochondrial protein complexes using sucrose density gradients after solubilization with n-dodecyl-β-D-maltoside; (c) 1D electrophoresis of the sucrose gradient fractions; (d) high-throughput proteomics using robotic gel band excision, in-gel digestion, MALDI target spotting and automated spectral acquisition; and (e) protein identification from mixtures of tryptic peptides by high-precision peptide mass fingerprinting. Using this approach, we rapidly identified 82 bona fide or potential mitochondrial proteins, 40 of which have not been previously reported using 2D PAGE techniques. These proteins include small complex I and complex IV subunits, as well as very basic and hydrophobic transmembrane proteins such as the adenine nucleotide translocase that are not recovered in 2D gels. The technique described here should also be useful for the identification of new protein-protein associations as exemplified by the validation of a recently discovered complex that involves proteins belonging to the prohibitin family. Keywords: MALDI-MS • mass spectrometry • membrane proteins • mitochondrial proteome • peptide mass fingerprinting • subcellular fractionation • sucrose gradient density centrifugation
Introduction In contrast to the formidable task of mapping entire proteomes of higher organisms, or even selected vertebrate organs, proteome analysis of subcellular organelles should present a more tractable approach.1 Mitochondria are a particularly attractive system in this regard. Their eubacteriotic origins manifest as a proteome comparable in size to those of many microorganisms. There are only 13 polypeptides encoded by human mitochondrial DNA, all of which constitute subunits of protein complexes that comprise the electron transport chain. However, there have been estimates of 1000-2000 proteins that are encoded in the nucleus and which undergo specific transport mechanisms through the cytosol and are targeted to the mitochondria.2 In fact, many proteins are localized, or targeted under specific conditions, to more than one organelle. For example, the GRIM 19 protein acts both as a complex I subunit and as a regulator of cell death.3 The interplay between the mitochondrial and nuclear genomes is vital for all eukaryotic life. In addition to providing the chemical * To whom correspondence should be addressed. Fax: (858) 509-5700. E-mail:
[email protected]. † MitoKor. ‡ Buck Institute. § University of Oregon. 10.1021/pr025533g CCC: $22.00
2002 American Chemical Society
energy that power cellular processes, mitochondria play important roles in metabolic signaling and cell death pathways. Evidence continues to mount that mitochondrial dysfunction is associated with many degenerative and metabolic diseases.4,5 To understand the interplay between the mitochondrial and nuclear genomes, some laboratories have focused on identifying proteins of small and large mitochondrial ribosomal subunits to compare and contrast the protein synthesizing machinery of eukaryotic and prokaryotic organisms.6-9 Other laboratories have focused on identifying mitochondrial proteins that may be important in apoptotic pathways. Patterson and co-workers studied proteins released from mitochondria during opening of the permeability transition pore complex (PTPC), a key step during apoptosis.10 Using LC/MS/MS, they identified 97 proteins or predicted proteins from expressed sequence tags that were released from mouse liver mitochondria after the pore opened. Very recently, Loo and co-workers identified 16 proteins released from mouse liver mitochondria treated with the apoptosis-inducing truncated form of Bid in a MALDIPSD study.11 In a global approach, several laboratories have undertaken large-scale mapping of entire mitochondrial proteomes. All of these latter studies have ultimately relied on obtaining highresolution 2D gel maps of mitochondrial proteins from which Journal of Proteome Research 2002, 1, 451-458
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Published on Web 08/23/2002
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Table 1. Distribution of Proteins within Complexes of the Mitochondrial Electron Transport Chain in Sucrose Density Gradients complex
protein
MW (kDa)
NCBI ACC no.
matches
MOWSEa
refb
sucrose fr.
I ND 13k NDUFS6 I ND 19k NDUFB8 I ND 19k NDUFA8 I ND 22k I ND 24k I ND 30k I ND 39k I ND 42k I ND 49k I ND 51k I ND 75k I ND B8 I ND B14 I ND B14.5a I ND B14.5b I ND B15 I ND B17 I ND B17.2 I ND B22
NADH dehydrog Fe-S protein 6 NADH dehydrog 1 β, sub 8 NADH dehydrog 1 R, sub 8 NADH dehydrog 1 β, sub 10 NADH dehydrog flavoprotein 2 NADH dehydrog Fe-S protein 3 NADH dehydrog 1 R, sub 9 NADH dehydrog 1 R, sub 10 NADH dehydrog Fe-S protein 2 NADH dehydrog flavoprotein 1 NADH dehydrog Fe-S protein 1 NADH dehydrog 1 R, sub 2 NADH dehydrog 1 R, sub 6 NADH dehydrog 1 R, sub 7 NADH dehydrog 1 unknown, sub 2 NADH dehydrog 1 β, sub 4 NADH dehydrog 1 β, sub 6 13kDa differentiation-associated protein NADH dehydrog 1 β, sub 9
13.6 21.7 20.1 20.7 27.3 30.2 42.5 40.7 52.5 50.9 79.4 10.9 15.1 12.6 14.2 15.2 15.4 17.1 21.8
4758792 4826854 14744612 4758774 10835025 11436882 6681764 4758768 4758786 6005816 13637608 4505355 11418201 4826850 14767065 11432336 14742250 10092657 14740729
6 5 6 13 9 9 16 10 15 13 23 5 5 7 7 8 6 5 11
369 (2020) 296 56700 (93400) 44700 1.13e + 009 7780 3.89e + 008 5.88e + 008 1.51e + 009 686 231 1960 263 53900 188 (2910) 11200
3 4 3 3, 4 3 3, P 3,4 6 2-4 2-4 2-4 3 3 3 3 2-4 3 3 3, 4
II
72.7
4759080
27
3.24e + 014
6, 7
II
succinate dehydrogenase sub A flavoprotein succinate dehydrogenase sub. B FeS
31.6
9257242
6
2930
6
III III III III III
ubiquinol-cyt c reductase binding protein ubiquinol-cyt c reductase core I ubiquinol-cyt c reductase core I ubiquinol-cyt c reductase Rieske FeS ubiquinol-cyt c reductase (cytochrome c1)
13.5 52.6 48.4 32.7 25.7
14743079 4507841 14775827 5174743 181238
6 18 20 6 6
1380 3.05e + 012 1.09e + 010 2110 784
4, 5 4, 5 4-6 5,6 4-6
IV IV IV IV IV
cytochrome c oxidase subunit IV cytochrome c oxidase subunit Va cytochrome c oxidase subunit Vb cytochrome c oxidase subunit VIb cytochrome c oxidase subunit VIc
19.6 16.8 13.7 10.2 8.8
4502981 4758038 16160063 4502985 4758040
16 4 6 5 4
2.03e + 008 527 723 12800 31.5
5,6 5 5,6 5-7 6
V V V V V V V
ATPase R ATPase β ATPase γ ATPase F0 subunit b ATPase F1F0 subunit d ATPase F0 subunit F6 ATPase oligomycin sensitivity con. protein
60.0 58.0 32.9 28.9 18.5 12.6 23.3
4757810 4502295 4885079 4502299 5453559 11526149 4502303
28 17 8 16 6 4 7
1.46e + 015 1.84e + 011 17700 2.93e + 009 9180 4400 7.46e + 005
4-8, P 4-8, P 4,5 5,6 5,6 5 4-7, P
14 13, 14 13
2, 12-14
2, 12, 13 13 12, 13
12
2, 13, 14 2, 12-14 2 2 2, 13, 14
a The best MOWSE score is shown for proteins found in multiple bands or fractions and is based on peptides matched using the Intellical algorithm within 15 ppm (unless in parentheses in which case there were no significant matches using Intellical but peptide matches are to high-intensity peaks in the MALDIMS within 100 ppm). b References to previous identifications of a given protein by 2D PAGE and PMF. Note: refs 2 and 14 dealt with nonhuman mitochondria and are cited if the nonhuman homologous protein was identified.
gel spots have been excised for protein identification by peptide mass fingerprinting (PMF)2,12-14 or immunostaining.15,16 One of the goals has been to produce a reference map to permit direct detection of differentially expressed gene products. However, the 2D gel approach suffers from several major drawbacks. Many membrane proteins are not observed because of precipitation and adsorption processes involving the IPG matrix when such strips are used for isoelectric focusing in the first dimension. Similarly, highly basic proteins are difficult to focus and are underrepresented in 2D gel maps. For example, none of the adenine nucleotide translocator (ANT) isoforms, which are both hydrophobic and highly basic, have been previously identified after 2D PAGE. Low molecular weight proteins also have proven elusive using the 2D PAGE approach, with few of the low molecular weight complex I and complex IV subunits being observed in previous studies. In addition, proteins must be visualized on a 2D gel to be analyzed. Comprehensive coverage of the entire gel area is not practical as it is for smaller 1D gels, although some interesting developments have recently been reported, that combine the transfer of proteins from 2D 452
Journal of Proteome Research • Vol. 1, No. 5, 2002
gels through trypsin-impregnated sheets to produce blots for subsequent MALDI-MS analysis and PMF.17 In the current study, we chose to adopt a different strategy of avoiding 2D gels, but of taking advantage of the resolving power of electrophoresis in a single dimension. This approach has been made possible by the optimization of sucrose gradients as our first “dimension” to separate intact mitochondrial complexes.15 Large protein complexes, such as complex I, have been found in the sucrose gradient fractions of highest density while free proteins tend to be found in the lighter sucrose fractions. In the second dimension, proteins within each sucrose fraction are separated by size with traditional 1D gel electrophoresis. By combining this tandem separation approach with extensive automation of the gel processing and protein identification stages, we have rapidly identified many proteins that have not been previously reported using 2D PAGE. This technique should allow discovery both of new mitochondrial proteins and new protein-protein associations based upon the sucrose gradient fraction in which a particular protein is identified. We provide one example of this in the prohibitin
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Toward the Human Heart Mitochondrial Proteome Table 2. Distribution of Other Proteins in Sucrose Density Gradients protein
MW (kDa)
NCBI ACC no.
matches
MOWSEa
sucrose fr.
refb
acetyl CoA acetyl transferase 1 acetyl CoA acyl transferase 2 aconitase 2 acyl CoA dehydrogenase (medium) acyl CoA dehydrogenase (very long chain) acyl CoA synthetase (long-chain) ANT 1 aralar aspartate amino transferase 2 precursor B-cell assoc. protein (D-prohibitin) citrate synthase precursor creatine kinase mitochondrial 2 cytochrome C δ-1-pyrroline-5-carboxylate dehydrogenase dihydrolipoamide dehydrogenase precursor dihydrolipoamide S-succinyltransferase electron-transferring flavoprotein dehydrogenase enoyl CoA hydratase 1, peroxisomal fumarate hydratase glutamate dehydrogenase 1 glyceraldehyde-3-phosphate dehydrogenase hexokinase 1 HSP20 (crystallin β B) HSP70 hydroxyacyl-CoA dehydrog/3-ketoacyl-CoA thiolase/enoylCo A hydratase: R subunit β subunit isocitrate dehydrogenase 2 (NADP+) malate dehydrogenase2 (NAD) methylcrotonoyl-coenzyme A carboxylase 2 (β) mitofilin monoamine oxidase A monoamine oxidase B nicotinamide nucleotide transhydrogenase oxoglutarate dehydrogenase E1 component peroxisomal long-chain acyl-CoA thioesterase phosphate carrier mitochondrial programmed cell death 8 prohibitin pyruvate dehydrogenase (lipoamide) R 1 succinate CoA ligase, R Tu translation elongation factor, mitochondrial unnamed protein product VDAC 1 VDAC 2
45.2 42.0 85.4 46.6 70.4 78.3 33.1 74.8 47.5 33.3 51.7 47.5 11.6 61.7 54.1 48.7 68.5 35.8 54.6 61.4 36.1 90.2 20.2 73.8 83.0
4557237 5174429 4501867 4557231 4557235 4503651 13647558 14747216 4504069 6005854 4758076 4502855 65436 14043187 14752229 14784523 4758312 11433007 14740547 4885281 7669492 11430299 4503057 12653415 4504325
7 18 22 8 12 14 17 14 20 13 13 15 5 7 11 10 4 12 8 7 10 4 5 18 19
1580 9.65e+011 9.12e+010 25200 71300 2.14e+005 1.07e+005 5.14e+006 4.24e+008 3020 121000 5.33e+008 103 5670 4970 1470 204 1.58e+005 593 1630 17000 141 109 1.7e+006 5.95e+012
6, 7 6 6-8 4, 6, 7 3, 5, 6 P 7 5-8, P 7 7 P, 3 6-8 5-7 9 1 2, 7 2 7 6 6 6 6 6 P 5, 7 2-6
14 14 12, 14 2, 12-14 2, 14 14
51.3 50.9 35.5 61.3 83.7 59.7 58.8 99.7 116.0 46.3 40.1 66.9 29.8 43.3 35.1 49.5 51.0 30.7 30.4
14730794 4504575 14782063 14721575 14732789 4557735 13631037 1141669 13627252 13375614 6031192 4757732 4505773 14762207 11321581 4507733 7022134 16171820 14736764
13 10 15 7 10 13 19 11 23 6 6 12 7 12 5 7 5 11 6
9540 31300 4.12e+007 114 1470 43300 1.17e+006 11700 1.89e+008 163 (8860) (38100) (3480) 4200 1690 7670 146 7580 238
2-6 7, 8 7, 8 7 2, 6 7 6, 7 6 2, 3 8 2, 4 7, 8 P 2 8 P 2 6, 7 5
13, 14 12, 14 13, 14
13 13, 14 13, 14 13 12 12, 14 13, 14 12, 13 2, 13, 14 12-14 12, 14 13 12, 14 14
13
13 2, 14 13 13, 14 13, 14
a The best MOWSE score is shown for proteins found in multiple bands or fractions and is based on peptides matched using the Intellical algorithm within 15 ppm (unless in parentheses in which case there were no significant matches using Intellical but peptide matches are to high-intensity peaks in the MALDIMS within 100 ppm). b References to previous identifications of a given protein by 2D PAGE and PMF. Note: References 2 and 14 dealt with nonhuman mitochondria and are cited if the nonhuman homologous protein was identified.
family of proteins, which are low molecular weight but which are found in a dense sucrose fraction, consistent with their newly discovered role as a molecular chaperone complex.18
Results and Discussion To our knowledge, the current study represents the fourth major effort to elucidate the mammalian mitochondrial proteome using high-resolution mass spectrometry as the final stage for protein identification.2,12-14 All of the previous reports have employed varying degrees of organellar fractionation. The most recent and comprehensive report identified 192 “gene products” from rat liver mitochondrial proteins isolated by successive centrifugation steps at 10000g and 100000g and then separated by six 2D PAGE gels with IPG strips spanning three different pH ranges.14 This exhaustive effort may represent the practical limits of the 2D gel approach toward the elucidation of the mitochondrial proteome. Furthermore, we note that the 192 gene products include apparent contaminants: e.g., serum albumin, actin, myosin (two entries), tropomyosin (two entries), lamin and keratin (two entries), as well as multiple entries for the same protein, e.g., sacrosine dehydrogenase (two entries:
identical accession numbers) and mitochondrial isocitrate dehydrogenase (two entries: bovine and macaque).14 In the current study, we have taken more extensive measures to exclude protein contaminants (see below), and we have listed only unique protein identifications in Tables 1 and 2. We also note that the authors of this 2D PAGE study were restricted by a limited protein database for rat compared with those for human and mouse, which contain four to five times as many entries. Integral to the approach we have taken is the isolation of highly purified mitochondria. We obtained human heart mitochondria from a commercial source and then further purified them by metrizamide gradient centrifugation. The purity of these mitochondria was assessed by Western analysis using antibodies directed against β-actin, dynamin II, KDEL, and LAMP to detect contamination due to cytoplasm, plasma membrane, ER, and lysosomes, respectively. Prior to metrizamide gradient purification, the mitochondria had no detectable dynamin II, KDEL, or LAMP. Actin was detected by Western analysis in the mitochondrial preparation, but not after metrizamide gradient purification (data not shown). The mitoJournal of Proteome Research • Vol. 1, No. 5, 2002 453
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Figure 2. MALDI mass spectrum of COX VIb (not previously identified using 2D PAGE). Peaks matching COX VIb peptides within 20 ppm (before recalibration using Intellical) are indicated by asterisks. T ) trypsin. Other abbreviations per Figure 1.
Figure 1. (a) Plot of western blot intensities for antibodies against selected subunits of protein complexes in the electron-transport chain for mitochondrial sucrose density fractions subjected to 1D PAGE (see the Experimental Section) and (b) corresponding Coomassie stained gel. ANT ) adenine nucleotide translocase. β ) ATP synthase β subunit. COX VIb ) cytochrome c oxidase subunit VIb. γ ) ATP synthase γ subunit. eCoA ) enoyl-CoA hydratase. vd ) voltage-dependent anion channel 1.
chondria were solubilized using the detergent n-dodecyl-β-Dmaltoside before application to the sucrose gradient.15 The integrity of mitochondrial protein complexes was assessed using a cocktail of monoclonal antibodies directed against components of the electron transport chain; NDUFS2, 70 kD subunit of complex II, core I of complex III, cytochrome c oxidase subunit IV (COX IV) of complex IV, and ATP synthase R of complex V. In Figure 1a, we have plotted the Western blot intensities for these selected subunits and found that the distribution of the complexes is slightly different from that reported by Hanson et al.,15 who used similar gels prepared from sucrose gradients of bovine heart mitochondria. The heaviest complex I peaks in concentration in fraction 3 of the gradient while the lighter complexes III, IV, and II peak in fractions 5, 6, and 7, respectively. With the exception of complex V, which continues to exhibit a broad distribution over four fractions, it appears that we have obtained, particularly at the bottom of the gradient, sharper distribution profiles and better resolution of the various complexes by employing higher volume steps (1 mL) than those used previously (0.5 mL).15 The protein profiles of the sucrose fractions, after being subjected to electrophoresis in a small format NuPAGE gel stained with colloidal Commassie blue, are shown in Figure 1b. In Tables 1 and 2, we have compiled the results of PMF analysis for two 1D gels: the small format gel in Figure 1b and a large format gel from which plugs were excised from 96 and 192 bands, respectively. In addition, we have included results for the corresponding gels run on the pellet (not shown). This insoluble material had been removed from the n-dodecyl-βD-maltoside treated mitochondria by centrifugation before the sucrose gradient fractionation and had been subsequently 454
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resolubilized using SDS before electrophoresis. The small format gel (Figure 1b) returned 44 unique protein identifications from the sucrose gradient fractions and the large format gel returned 49 unique protein identifications. These data sets were merged with the pellet data, redundant identifications were removed, and ultimately 82 unique proteins were identified, of which 40 have not been previously reported using 2D PAGE techniques. In general, we found that the large format gel had better resolution but protein concentrations within a given band tended to be more dilute. In contrast, the smaller format gel produced higher local protein concentrations within a given band but resolution of proteins was poorer. Our ability to make high-precision MALDI measurements compensated to a large extent for the diminished resolution in the smaller gel (see the Experimental Section). We were able to identify 19 of the complex I subunits that were distributed in lanes loaded with heavy sucrose fractions 2-4 (Table 1). Of these, the 39 kDa and 51 kDa subunits, and the 12 subunits below 30 kDa have not been previously reported using 2D PAGE strategies. Similarly, we identified five complex IV subunits distributed over gel lanes run for the lighter fractions 5-7. Of these, only one subunit has previously been reported using the 2D gel approach.12 A representative PMF for cytochrome c oxidase subunit VIb, a 10.2 kDa subunit of complex IV excised from the small gel in Figure 1b, is shown in Figure 2. A dominant protein found in fractions 5-6, as well as in the pellet, is the adenine nucleotide translocator (ANT isoform 1) (Figures 1b and 3). As well as being an extremely hydrophobic membrane protein, ANT 1 is very basic with a calculated pI of 9.8, thus making it difficult to focus by IEF. The level of the ANT protein found in this preparation, as judged by its intensity on Coomassie staining (Figure 1b), makes it conspicuous by its absence from all previous proteomic approaches that employ 2D PAGE. Similarly, the inner membrane-bound phosphate carrier, not previously reported on 2D gels, and the two membrane bound isoforms of the voltage dependent anion channels (VDACs), were readily identified in the current study. We identified only 2 proteins associated with the pyruvate dehydrogenase complex. These polypeptides were found in
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Toward the Human Heart Mitochondrial Proteome Table 3. Distribution of Apparent Contaminant Proteins from Mitochondrial Preparations protein
MW (kDa)
NCBI ACC no.
matches
MOWSEa
sucrose fr.
R-cardiac actin myosin, light polypeptide 6 lamin A/C histone 4 family dynamin 1 tubulin
49.2 42.0 16.9 65.1 11.3 97.4 48.6
1142319 4501883 11435115 5031875 4504321 4758182 11431691
9 6 4 8 6 8 5
(9700) (7060) 2700 8910 1810 1690 (1030)
2, 8, 9 P 6 P P 6, P 6, P
keratinb
a The best MOWSE score is shown for proteins found in multiple bands or fractions and is based on peptides matched using the Intellical algorithm within 15 ppm (unless in parentheses in which case there were no significant matches using Intellical but peptide matches are to high-intensity peaks in the MALDIMS within 100 ppm). b Peaks not included in keratin peak filter.
contaminants as described above.13 The few nonmitochondrial proteins that were observed by PMF are listed in Table 3 and appear to be mainly confined to the pellet. Although PMF is by no means quantitative, we note that the relatively low number of confident peptide matches for these proteins is consistent with their presence in small amounts as suggested by the Western analysis. Incidences of keratin contamination were rare, probably as a result of our avoidance of excessive sample handling through the use of robotics for gel processing. The presence of glyceraldehyde-3-phosphate dehydrogenase in this highly purified mitochondrial preparation, as well as in a previous report,13 is therefore very interesting. This normally cytosolic protein has been reported to play a role in apoptosis,19 most recently by accumulating in the nucleus as a consequence of signal transduction.20 In light of our results, it is now interesting to speculate on a mitochondrial role for this protein in cell death. Figure 3. MALDI mass spectrum of ANT 1 (not previously identified using 2D PAGE). Peaks matching ANT peptides with 20 ppm (before recalibration using Intellicala¨ ) are indicated by asterisks.
sucrose fraction 2, consistent with this matrix protein’s extremely large size (108 subunits), and they included the dihydrolipoamide dehydrogenase precursor and pyruvate dehydrogenase (lipoamide) R 1. Interestingly, some dihydrolipoamide dehydrogenase was also found in lighter fraction 7, suggesting some dissociation or that an equilibrium exists between a free and bound form of the protein. We also identified two subunits of the 2-oxoglutarate dehydrogenase complex including the E2 subunit; dihydrolipoamide S-succinyltransferase, in sucrose fraction 2, as well as the E1 dehydrogenase (lipoamide) component which, at 116 kDa, was the largest protein identified in our study. On the smaller end of the size spectrum, we identified the low molecular weight (11.6 kDa) cytochrome c, a protein that has not been identified in 2D gel maps, in the light sucrose fraction 9. We have also observed that some proteins, which were readily identified on 2D gel maps, did not run well on the sucrose gradients. These proteins, such as prohibitin and D-prohibitin (also known as a B-cell associated protein), were found primarily localized in the mitochondrial pellet. The latter protein was also detected in the heavy sucrose fraction 3. This result is consistent with the finding that these proteins form a larger complex that acts as a molecular chaperone for respiratory complex assembly.18 Few nonmitochondrial proteins were identified in the current investigation by PMF. We attribute this lack to the use of the metrizamide gradient procedure for purifying mitochondria which led to scrupulous removal of other extraorganellar
In summary, we have developed a different and complementary strategy for mitochondrial proteome analysis that takes advantage of the ability to produce highly purified mitochondria and to separate intact active protein complexes. In the future, we plan to analyze each sucrose gradient fraction by LC/MS/MS after electrophoresis and also to employ the multidimensional protein identification method described by Link et al., which offers a high-throughput non-gel-based approach for protein identification.21 Further, we plan to use monoclonal antibodies to remove specific high abundance proteins and complexes that may mask undiscovered mitochondrial proteins of low copy number. These latter proteins will be subject to mitochondrial protein motif analysis and prediction programs to ultimately direct future cloning and functional efforts to elucidate possible links between new proteins, normal mitochondrial function, and disease.
Experimental Section Preparation of Mitochondria. Human heart mitochondria were obtained from Analytical Biological Services, Inc. (Wilmington, DE) using normal ventricle muscle removed postmortem from a 62 year old female, who died as the result of intracranial bleeding, and were further purified by metrizamide gradient centrifugation.22 Mitochondria (10 mg wet weight) were resuspended in MSHE (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, 1 mM EGTA plus a Complete protease inhibitor cocktail tablet (Roche, Indianapolis, IN)) and loaded onto a 35%/17% metrizamide gradient in 6% Percoll. Gradients were centrifuged at 4 °C for 45 min and at 19000 rpm in a SW40 rotor. The heavy mitochondrial fraction was collected from the 35/17% interface, diluted in MSHE before pelleting at 12000g for 10 min, and resuspended in MSHE. Protein concentrations were determined using the BioRad DC protein assay. The purity Journal of Proteome Research • Vol. 1, No. 5, 2002 455
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Table 4. Protein Prospector Search Parameters and Results for Peptide Peaks in the MALDI Spectrum in Figure 4 (Human nr Database) before (a) and after (b) Application of the Intellical Algorithm (a) min no. peptides to match
peptide mass tolerance (()
peptide masses are
5
100 000 ppm
monoisotpoic
digest used
max no. missed cleavages
cysteines modified by
trypsin
1
carbamidomethylation
rank
MOWSE score
no. (%) masses matched
protein MW (Da) pI
species
nr. human acc no.
1 2 3
1.81e+003 1.08e+003 943
8/50 (16%) 5/50 (10%) 7/50 (14%)
30772.7/8.62 47232.3/4.87 84157.6/5.79
Homo sapiens Homo sapiens Homo sapiens
4507879 4504921 11432236
4
804
5/50 (10%)
35816.4/8.16
Homo sapiens
11433007
peptide N terminus
peptide C terminus
input no. peptide masses
hydrogen (H)
free acid (OH)
50
protein name
voltage-dependent anion channel 1 keratin, hair, acidic, 1 similar to neruoblastoma-amplified protein (H. sapiens) enoyl coenzyme A hydratease 1, peroxisomal
(b) min no. peptides to match
peptide mass tolerance (()
peptide masses are
4
15 000 ppm
monoisotpoic
rank
MOWSE score
digest used
max no. missed cleavages
cysteines modified by
trypsin
1
carbamidomethylation
no. (%) masses matched
protein MW (Da) pI
species
nr. human acc no.
1 2
804 304
5/50 (10%) 5/50 (10%)
35816.4/8.16 51671.6/5.55
Homo sapiens Homo sapiens
11433007 11435090
3 4
77.5 60.1
5/50 (10%) 5/50 (10%)
30772.7/8.62 32881.1/9.31
Homo sapiens Homo sapiens
4507879 4885079
of the mitochondria was assessed by Western analysis using antisera directed against actin (Abcam, Cambridge, UK), dynamin II (Transduction Labs, Lexington, KY), KDEL, and LAMP1 (Stressgen, Victoria, BC, Canada) to detect contamination due to cytoplasm, plasma membrane, ER, and lysosomes, respectively. The integrity of the mitochondria was assessed by Western analysis using a cocktail of antibodies directed against components of the electron transport chain; NDUFS2, 70 kD subunit of complex II, core I of complex III, COX IV, and ATP synthase R; all from Molecular Probes (Eugene, OR). Sucrose Gradient Density Centrifugation. Metrizamidepurified mitochondria (4 mg) were resuspended in MSHE plus protease inhibitors and solubilized with 1% n-dodecyl-β-Dmaltoside for 25 min on ice with frequent vortexing. Samples were centrifuged at 4 °C and at 14000 rpm for 20 min. The pellet was frozen by immersion in liquid nitrogen and stored at -80 °C. The supernatant was subjected to sucrose gradient centrifugation.15 The gradient consisted of 1 mL step-fractions of 35, 32.5, 30, 27.5, 25, 22.5, 20, 17.5, 15, and 10% sucrose in 10 mM Tris, pH 7.5/1 mM EDTA/0.05% n-dodecyl-β-D-maltoside, plus protease inhibitors). The solubilized mitochondria were loaded onto the gradient in 5% sucrose and centrifuged at 38000 rpm, and at 4 °C for 16.5 h in a SW40 rotor. The gradient was collected from the bottom in 1 mL fractions. Samples were frozen by immersion in liquid nitrogen and stored at -80 °C. Separation of proteins across the gradient was assessed by 4-12% NuPAGE in MES buffer (Invitrogen, Carlsbad, CA) followed by staining with SimplyBlue Safe Stain (Invitrogen) or Western analysis using the cocktail of antibodies directed against components of the electron transport chain. Aliquots 456
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peptide N terminus
peptide C terminus
input no. peptide masses
hydrogen (H)
free acid (OH)
50
protein name
enoyl coenzyme A hydratease 1, peroxisomal ATPsynthase, H+ transporting, mitochondrial F1 complex, β polypeptide voltage-dependent anion channel 1 ATPsynthase, H+ transporting, mitochondrial F1 complex, γ polypeptide
(0.5 mL) of the gradient fractions were concentrated in Microcon YM-3 centrifugal concentrators (Millipore, Bedford, MA) and brought up to a final volume of 100 µL with NuPage-LDS sample buffer (Invitrogen). For protein analysis by MALDI, a NuPage gel was loaded with 20 µL aliqouts of the concentrated gradient fractions and run. Larger format 8-16% Tris-HCl gradient gels were purchased from BioRad (Hercules, CA). The remainder of each concentrated sample (80 µL) was separated on a BioRad Protean II gel apparatus in Tris/glycine/SDS buffer per the manufacturer’s instructions. Quantitation of the electrontransport chain complexes across the gradient was performed on images captured on a Fluor-S MultiImager (BioRad, Hercules, CA) and analyzed using Quantity One software (BioRad). Gel Processing and Sample Handling. Electrophoretic gels were placed on the platform of a ProteomeWorks Robotic Imager and Spot Cutter (BioRad). After images were acquired, gel bands were selected for robotic excision and their coordinates on the gel image were saved to a file using PDQuest software (BioRad). For each gel band, plugs were excised with a 14 gauge stainless steel circular cutting head that rapidly rotates, drilling into the gel. The cutting head then transfers the plugs to a 96-well microtiter plate and the PDQuest software annotates the bands on the gel image with a unique well number. One 96-well plate was filled with plugs from the small format gel and two from the large format gel. Gel plugs were subjected to automated destaining, reduction, alkylation, and proteolysis using a ProGest digestion robot (Genomic Solutions, Inc.). This robot delivers aliquots of the reagents and then purges them from the plugs with a stream of nitrogen through holes pierced in the bottom of a custom 96 deep well conical
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Toward the Human Heart Mitochondrial Proteome Table 5. Detailed Results and Mass Accuracies for the Proteins Identified in Table 4
A. 5/50 matches (10%); 35816.4 Da, pI ) 8.16; acc no. 11433007; Homo sapiens; enoyl coenzyme A hydratase 1, peroxisomal m/z submitted
MH+ matched
δ, ppm
start
end
1298.6542 1428.6569 1454.8306 1542.8263 1731.9272 1731.9272
1298.6381 1428.6615 1454.8344 1542.8128 1731.9394 1731.9203
12.3728 -3.2457 -2.6081 8.7627 -7.0100 4.0158
149 88 66 197 215 251
158 98 77 211 230 267
peptide sequence
(R)YQETFNVIER(C) (R)EMVECFNKISR(D) (K)HVLHVQLNRPNK(R) (K)EVDVGLAADVGTLQR(L) (K)VIGNQSLVNELAFTAR(K) (K)EVMLDAALALAAEISSK(S)
modifications
1Met-ox
B. 5/50 matches (10%); 51671.6 Da, pI ) 5.55; acc no. 11435090; Homo sapiens; ATP synthase; H+ transporting; mitochondrial F1 complex; β polypeptide m/z submitted
MH+ matched
δ, ppm
start
end
1406.6879 1435.7636 1617.8089 1650.9120 1919.0695
1406.6817 1435.7545 1617.8059 1650.9179 1919.0966
4.4439 6.3356 1.8679 -3.5517 -14.1403
222 307 261 91 121
235 320 275 105 139
peptide sequence
(K)AHGGYSVFAGVGER(T) (R)FTQAGSEVSALLGR(I) (K)VALVYGQMNEPPGAR(A) (R)LVLEVAQHLGESTVR(T) (K)VLDSGAPIKIPVGPETLGR(I)
modifications
1Met-ox
C. 5/50 matches (10%); 30772.7 Da, pI ) 8.62; acc no. 4507879; Homo sapiens; voltage-dependent anion channel 1 m/z submitted
MH+ matched
δ, ppm
start
end
1414.6450 1528.7687 1693.8150 1698.8624 2175.9795
1414.6313 1528.7647 1693.8226 1698.8703 2175.9803
9.6783 2.5890 -4.4818 -4.6354 -0.3629
225 97 62 94 121
236 110 74 109 139
peptide sequence
(K)YQIDPDACFSAK(V) (K)LTFDSSFSPNTGKK(N) (K)YRWTEYGLTFTEK(W) (R)GLKLTFDSSFSPNTGK(K) (R)EHINLGCDMDFDIAGPSIR(G)
modifications
1Met-ox
D. 5/50 matches (10%); 32881.1 Da, pI ) 9.31; acc no. 4885079; Homo sapiens; ATP synthase, H+ transporting, mitochondrial F1 complex, γ polypeptide 1; ATP synthase, H+ transporting, mitochondrial F1 complex, γ polypeptide-like 1 m/z submitted
MH+ matched
δ, ppm
start
end
1292.6779 1326.7472 1345.7616 1733.8965 1768.8853
1292.6639 1326.7309 1345.7513 1733.8975 1768.8904
10.8216 12.2559 7.5991 -0.5833 -2.885
144 68 127 144 263
154 79 138 158 277
plate (Genomic Solutions, Inc.). Plugs were destained using two cycles of redehydration and dehydration with 25 mM ammonium bicarbonate and acetonitrile, which were successively added in 50 µL aliquots per plug. Reduction with 40 µL of 10 mM DTT per gel plug was performed at 65 °C for 10 min followed by treatment with 30 µL of 100 mM iodoacetamide at room temperature. After another two cycles of redehydration and dehydration with 25 mM ammonium bicarbonate and acetonitrile as described above, successive aliquots of trypsin (56 ng) in 10 µL of 1 mM acetic acid and 15 µL of 25 mM ammonium bicarbonate were added to each plug. Digestion was allowed to proceed at 37 °C for 4 h and then was terminated by addition of a 7 µL aliquot of formic acid. The digests were then transferred from plugs into another 96-well receiving plate by purging the liquid through the holes in the wells of the top plate with a stream of nitrogen. This plate was directly placed in a Symbiot (Applied Biosystems) robotic MALDI target spotter, and 0.5 µL aliquots were spotted on a 2 × 96 well PS1 MALDI target along with a 0.3 µL aliquot of R-hydroxycinnamic acid matrix in 50% ACN, 0.1% TFA. Between each row of sample spots, calibrant (Des Arg1 bradykinin, Mr 904.4681;1 angiotensin 1, 1296.6853; Glu1-fibrinopeptide B, 1570.6774; neurotensin, 1672.9175) was spotted for close external calibration between each successive MALDI spectrum. Acquisition and Analysis of Mass Spectra. MALDI spectra were acquired on a Voyager DE-STR under the following conditions: positive reflectron mode with delayed extraction, (1) Isotopic C-12 masses.
peptide sequence
(R)THSDQFLVAFK(E) (R)IYGLGSLALYEK(A) (K)EVMLGIGDKIR(G) (R)THSDQFLVAFKEVGR(K) (K)NASEMIDKLTLTFNR(T)
modifications
1Met-ox 1Met-ox
accelerating voltage 20 kV, grid voltage 65%, mirror voltage ratio 1.12, extraction delay time 125 ns, and low mass gate 500 Da. Spectral acquisition was automated using a spiral search pattern with saved spectra being the average of three successful acquisitions from 400 laser shots at 20 Hz repetition rate in the m/z 850-3000 range with a minimum intensity of 750 counts in the m/z 1000-3000 range. Peptide mass fingerprints from baseline-corrected, noise-filtered de-isotoped spectra were filtered to remove autolytic trypsin and most keratin peaks and then analyzed using the program Protein Prospector.23 A limitation of our technique compared with 2D gels is that every 1D band that was analyzed consisted of mixtures of at least two proteins. This is true even before consideration of the presence of isoforms of a single protein (e.g., due to differential phosphorylation) that can be typically resolved on 2D gels. Nevertheless, the high mass accuracy of the MALDITOF instrument, estimated to be within (10-50 ppm, made it possible to identify the individual protein components within mixtures. Additional confidence could be gained by using the Intellical algorithm of the Proteomics Solution 1 (PS1) software (Applied Biosystems), which takes into account the high precision in MALDI-TOF measurements by internally recalibrating spectral peaks against peptide masses for highconfidence protein identifications. In Table 4, the voltagedependent anion channel was identified with the highest confidence from the MALDI-TOF spectrum in Figure 4 by a total of eight peptides ranging in accuracy from -31 to -94 ppm compared with the calculated values (data not shown). Journal of Proteome Research • Vol. 1, No. 5, 2002 457
research articles
Taylor et al.
somal enoyl CoA hydratase is one of the few examples of extraorganellar contaminants in our preparation that is clearly distinguished from the highly homologous mitochondrial 3,5δ-2,4-dienoyl-CoA isomerase by the last two tryptic peptides listed in Table 5A. Nevertheless, we list this protein in Table 2 since a mitochondrial association for the protein cannot be ruled out without further study. Note Added after ASAP. The version of this paper posted August 23, 2002, contained the wrong ref 22. The version with the correct reference was posted on August 28, 2002.
References
Figure 4. MALDI mass spectrum for a single band excised from a 1D gel with peptides from protein components indicated. K ) keratin. Other abbreviations per Figure 1. The peak masses are listed in Table 5.
For comparison, keratin and the neuroblastoma-amplified protein’s peptide hits deviated -93 to +87 ppm from the calculated values, indicating low precision in contrast with peroxisomal enoyl CoA hydratase peptides values that were within -60 and -97 ppm of the predicted values. Using the Intellical algorithm, four different protein components could be identified from Figure 4 (Tables 4 and 5). Because these searches are more rigorous, having a precision as well as an accuracy component, more peptides are eliminated and MOWSE scores24 are typically lower. The proteins included two which were identified in our more tolerant search: peroxisomal enoyl CoA hydratase and the voltage-dependent anion channel, and two which were not: ATP synthase β and γ subunits. Keratin and the neuroblastoma-amplified protein were no longer confident identifications under these searching conditions, although major peaks ascribed to keratin could be observed in this spectrum. This result indicates one of the limitations of the Intellical algorithm searches: that they can be too rigorous and legitimate hits can be lost or drop dramatically in confidence. Thus, when evaluating a hit from MALDI data, we routinely compared MOWSE scores for searches with and without the Intellical algorithm, monitored the relative intensity of the spectral peaks ascribed to a particular protein, and finally, sought correlation between the molecular weight of the identified protein with the position of the gel band that was excised. In the case of ATP synthase β subunit, a short form or degradation product was clearly identified because the molecular weight of the protein is 20 kDa higher than the other three proteins identified in this gel sample (the tryptic peptides identified from ATP synthase β subunit encompass a 25 kDa region of the protein sequence). Finally, we note that peroxi-
458
Journal of Proteome Research • Vol. 1, No. 5, 2002
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PR025533G
